Genetic sequences in eukaryotic cells that code for proteins are transcribed by class II RNA polymerases. This transcription is tightly regulated and, hence, is a major regulatory process for cellular growth and development. While RNA polymerase II and the structure of the genes it transcribes have been the subject of intensive study, it has been difficult to obtain detailed information as to the biochemical processes involved in nuclear transcription and its regulation. In particular, it has not yet been possible to assemble an in vitro transcription system with RNA polymerase and other defined components, in which transcription occurs at a rate, and with a specificity, characteristic of the in vivo process. Such reconstituted systems are essential if one is to carry out biochemical studies on the transcription process and understand the molecular action of external regulatory factors. The major goals of the proposed research are: (1) Studies on the effect of template and transcript structure on RNA chain elongation and possible termination by purified mammalian RNA polymerase II and mapping of the structural features of the ternary transcription complex. These will employ an efficient system we have developed for transcription of specific DNA sequences by purified mammalian RNA polymerase II. (2) Identification and characterization of cellular factors that may be involved in efficient elongation of RNA chains on DNA and chromatin templates. (3) Development of a system for in vitro transcription of specific DNA sequences by purified RNA polymerase II together with accessory factors that mimics the process found in vivo.